Methods for producing sulphurous fine chemicals by fermentation using metH-coding cornyeform bacteria

ABSTRACT

The invention relates to methods for the fermentative production of sulfur-containing fine chemicals, in particular L-methionine, by using bacteria which express a nucleotide sequence coding for a methionine synthase (metH) gene.

RELATED APPLICATION

The current application claims priority from the following InternationalPatent Application filed pursuant to Patent Cooperation Treaty (PCT) onApr. 16, 2003, designating the United States, which claims priority fromGerman Patent Application S/N 10217058.4 DE filed on Apr. 17, 2002. TheInternational Patent Application is assigned International ApplicationNumber, PCT/EP03/04010 and names all the same inventors as thisapplication: Ser. No. 10/511,302 entitled Methods for ProducingSulphurous Fine Chemicals by Fermentation Using Meth-Coding CornyeformBacteria. The International Patent Application was published in Germanon Oct. 23, 2003, and assigned International Publication Number: WO2003/087386.

TECHNICAL FIELD OF INVENTION

The invention relates to a method for the fermentative production ofsulfur-containing fine chemicals, in particular L-methionine, by usingbacteria which express a nucleotide sequence coding for a methioninesynthase (metH) gene.

BACKGROUND

Sulfur-containing fine chemicals such as, for example, methionine,homocysteine, S-adenosylmethionine, glutathione, cysteine, biotin,thiamine, lipoic acid are produced in cells via natural metabolicprocesses and are used in many branches of industry, including the food,animal feed, cosmetics and pharmaceutical industries. These substanceswhich are collectively referred to “sulfur-containing fine chemicals”include organic acids, both proteinogenic and nonproteinogenic aminoacids, vitamins and cofactors. They are most expediently produced on alarge scale by means of cultivating bacteria which have been developedin order to produce and secrete large amounts of the substance desiredin each case. Organisms which are particularly suitable for this purposeare coryneform bacteria, Gram-positive nonpathogenic bacteria.

It is known that amino acids are produced by fermentation of strains ofcoryneform bacteria, in particular Corynebacterium glutamicum. Due tothe great importance, the production processes are constantly improved.Process improvements can relate to measures regarding technical aspectsof the fermentation, such as, for example, stirring and oxygen supply,or to the nutrient media composition such as, for example, sugarconcentration during fermentation or to the work-up to give the product,for example by ion exchange chromatography, or to the intrinsicperformance properties of the microorganism itself.

A number of mutant strains which produce an assortment of desirablecompounds from the group of sulfur-containing fine chemicals have beendeveloped via strain selection. The performance properties of saidmicroorganisms are improved with respect to the production of aparticular molecule by applying methods of mutagenesis, selection andmutant selection. However, this is a time-consuming and difficultprocess. In this way strains are obtained, for example, which areresistant to antimetabolites or inhibitors such as, for example, themethionine analogs α-methylmethionine, ethionine, norleucine,n-acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5-heprenoiticacid, selenomethionine, methioninesulfoximine, methoxine,1-aminocyclopentanecarboxylic acid or which are auxotrophic formetabolites important for regulation and which produce sulfur-containingfine chemicals such as, for example, L-methionine.

Methods of recombinant DNA technology have also been used for some yearsto improve Corynebacterium strains producing L-amino acids by amplifyingindividual amino-acid biosynthesis genes and investigating the effect onamino acid production.

WO-A-02/10209 describes a method for the fermentative production ofL-methionine using L-methionine-producing coryneform bacteria in whichat least the metH gene is overexpressed and also the coding metHsequence from C. glutamicum ATCC 13032.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel method forthe improved fermentative production of sulfur-containing finechemicals, in particular L-methionine.

We have found that this object is achieved by providing a method for thefermentative production of a sulfur-containing fine chemical, comprisingthe expression of a heterologous nucleotide sequence coding for aprotein with metH activity in a coryneform bacterium.

The invention firstly relates to a method for the fermentativeproduction of at least one sulfur-containing fine chemical, whichcomprises the following steps:

-   -   a) fermentation of a coryneform bacteria culture producing the        desired sulfur-containing fine chemical, the coryneform bacteria        expressing at least one heterologous nucleotide sequence which        codes for a protein with methionine synthase (metH) activity;    -   b) concentration of the sulfur-containing fine chemical in the        medium or in the bacterial cells, and    -   c) isolation of the sulfur-containing fine chemical, which        preferably comprises L-methionine.

The above heterologous metH-encoding nucleotide sequence is preferablyless than 70% homologous to the metH-encoding sequence fromCorynebacterium glutamicum ATCC 13032. The metH-encoding sequence isderived preferably from any of the following organisms of list I:

List I Streptomyces coelicolor ATCC 10147 Anabaena sp. ATCC 27892Synechocystis sp. ATCC 27184 Prochlorococcus marinus PCC 7118 Thermusthermophilus ATCC 27634 Bacillus halodurans ATCC 21591 Bacillusstearothermophilus ATCC 12980 Vibrio cholerae ATCC 39315 Sinorhizobiummeliloti ATCC 4399 Escherichia coli K12 ATCC 55151 Salmonellatyphimurium ATCC 15277 Salmonella typhi ATCC 12839 Pseudomonasfluorescens ATCC 13525 Pseudomonas aeruginosa ATCC 17933 Nitrosomonaseuropeae ATCC 19718 Bordetella pertussis ATCC 9797 Clorobium tepidumATCC 49652 Deinococcus radiodurans ATCC 13939 Clostridium acetobutylicumATCC 824 Caulobacter crescentus ATCC 19089 Homo sapiens Vibrio fischeriATCC 33715 Agrobacterium tumefaciens str. C58 (Cereon) ATCC 33970Ralstonia solanacearum ATCC 25237 ATCC: American Type CultureCollection, Rockville, MD, USA PCC: Pasteur Culture Collection ofCyanobacteria. Paris France

The metH-encoding sequence used according to the invention preferablycomprises a coding sequence according to SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49 and 51 or a nucleotide sequence homologous thereto which codes for aprotein with metH activity.

Moreover, the metH-encoding sequence used according to the inventionpreferably codes for a protein with metH activity, said proteincomprising an amino acid sequence according to SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50 and 52 or an amino acid sequence homologous thereto whichrepresents a protein with metH activity.

The coding metH sequence is preferably a DNA or an RNA which can bereplicated in coryneform bacteria or is stably integrated into thechromosome.

According to a preferred embodiment, the method of the invention iscarried out by

a) using a bacterial strain transformed with a plasmid vector whichcarries at least one copy of the coding metH sequence under the controlof regulatory sequences or

b) using a strain in which the coding metH sequence has been integratedinto the bacterial chromosome.

Furthermore, preference is given to overexpressing the coding metHsequence for the fermentation.

It may also be desirable to ferment bacteria in which additionally atleast one further gene of the biosynthetic pathway of the desiredsulfur-containing fine chemical or of a biosynthetic or other metabolicpathway associated therewith has been amplified; and/or in which atleast one metabolic pathway, which reduces production of the desiredsulfur-containing fine chemical has, at least partially, been switchedoff.

It may also be desirable to ferment bacteria in which additionally theactivity of at least one further gene of the biosynthetic pathway of thedesired sulfur-containing fine chemical is not undesirably influenced bymetabolic metabolites.

Therefore, according to another embodiment of the method of theinvention, coryneform bacteria are fermented in which, at the same time,at least one of the genes selected from among

-   -   a) the aspartate kinase-encoding gene lysC,    -   b) the aspartate-semialdehyde dehydrogenase-encoding gene asd,    -   c) the glyceraldehyde-3-phosphate dehydrogenase-encoding gene        gap,    -   d) the 3-phosphoglycerate kinase-encoding gene pgk,    -   e) the pyruvate carboxylase-encoding gene pyc,    -   f) the triose phosphate isomerase-encoding gene tpi,    -   g) the homoserine O-acetyltransferase-encoding gene metA,    -   h) the cystathionine gamma-synthase-encoding gene metB,    -   i) the cystathionine gamma-lyase-encoding gene metC,    -   j) the serine hydroxymethyltransferase-encoding gene glyA,    -   k) the O-acrylhomoserine sulfhydrylase-encoding gene metY,    -   l) the methylene tetrahydrofolate reductase-encoding gene metF,    -   m) the phosphoserine aminotransferase-encoding gene serC,    -   n) the phosphoserine phosphatase-encoding gene serB,    -   o) the serine acetyl transferase-encoding gene cysE,    -   p) the homoserine dehydrogenase-encoding gene hom is        overexpressed.

According to another embodiment of the method of the invention,coryneform bacteria are fermented in which, at the same time, at leastone of the genes selected from among genes of the abovementioned groupa) to p) is mutated in particular in such a way that the activity of thecorresponding proteins is influenced by metabolic metabolites to asmaller extent, if at all, compared to nonmutated proteins and that inparticular the inventive production of the fine chemical is notadversely affected. Owing to the mutation, the protein may also havehigher activity (substrate conversion) and/or substrate specificity andthus enhance the production of the desired fine chemical.

According to another embodiment of the method of the invention,coryneform bacteria are fermented in which, at the same time, at leastone of the genes selected from among

-   -   q) the homoserine kinase-encoding gene thrB,    -   r) the threonine dehydratase-encoding gene ilvA,    -   s) the threonine synthase-encoding gene thrC,    -   t) the meso-diaminopimelate D-dehydrogenase-encoding gene ddh,    -   u) the phosphoenolpyruvate carboxykinase-encoding gene pck,    -   v) the glucose-6-phosphate 6-isomerase-encoding gene pgi,    -   w) the pyruvate oxidase-encoding gene poxB,    -   x) the dihydrodipicolinate synthase-encoding gene dapA,    -   y) the dihydrodipicolinate reductase-encoding gene dapB; or    -   z) the diaminopicolinate decarboxylase-encoding gene lysA is        attenuated, in particular by reducing the rate of expression of        the corresponding gene, or by expressing a protein having lower        activity (substrate conversion).

According to another embodiment of the method of the invention,coryneform bacteria are fermented in which, at the same time, at leastone of the genes of the above groups q) to z) is mutated in such a waythat the enzymic activity of the corresponding protein is partially orcompletely reduced.

Preference is given to using, in the method of the invention,microorganisms of the species Corynebacterium glutamicum.

In a further embodiment of the method, those microorganisms which areresistant to at least one methionine biosynthesis inhibitor areemployed. Such inhibitors are methionine analogs such asα-methylmethionine, ethionine, norleucine, N-acetylnorleucine,S-trifluoromethylhomocysteine, 2-amino-5-heprenoic acid,selenomethionine, methioninesulfoximine, methoxine and1-aminocyclopentanecarboxylic acid.

The invention further relates to a method for producing anL-methionine-containing animal feed additive from fermentation broths,which comprises the following steps:

-   -   a) culturing and fermentation of an L-methionine-producing        microorganism in a fermentation medium;    -   b) removal of water from the L-methionine-containing        fermentation broth;    -   c) removal of from 0 to 100% by weight of the biomass formed        during fermentation; and    -   d) drying of the fermentation broth obtained according to b)        and/or c), in order to obtain the animal feed additive in the        desired powder or granule form.

The invention likewise relates to the coding metH sequences isolatedfrom the above microorganisms for the first time, to the methioninesynthases encoded thereby and to the functional homologs of thesepolynucleotides and proteins, respectively.

In particular, the invention also relates to the expression constructsand microorganisms required for carrying out the above methods.

The invention therefore also relates to the following:

-   -   plasmid pCIS lysC thr311ile encoding lysC thr311ile or a        functional equivalent thereof, i.e. a lysC mutant with        comparable aspartate kinase activity which is increased over the        wild type;    -   a host organism transformed with plasmid pCIS lysC thr311ile, in        particular selected from among microorganisms of the genus        Corynebacterium, in particular of the species C. glutamicum,        such as transformed strain LU1479 lysC 311ile;    -   plasmid pC Phsdh metH Sc encoding Streptomyces coelicolor metH;    -   a host organism as defined above, transformed with a plasmid        encoding exogenous metH; in particular transformed with the        plasmid pC Phsdh metH Sc;    -   a host organism as defined above with resistance to at least one        methionine biosynthesis inhibitor such as the transformed strain        LU1479 lysC 311ile ET-16, optionally transformed with an        exogenous coding metH sequence, such as the transformed strain        LU1479 lysC311ile ET-16 pC Phsdh metH Sc.

DETAILED DESCRIPTION OF THE INVENTION

a) General Terms

Proteins with the biological activity of methionine synthase, also metHfor short (systematic name: 5-methyltetrahydrofolate homocysteinesS-methyltransferase; EC 2.1.1.13), refer to those proteins which arecapable of converting homocysteine to methionine and tetrahydrofolateusing the cofactors 5-methyltetrahydrofolate (MTHF), cobalamin (vitaminB12) and S-adenosylmethionine. While the cofactor5-methyltetrahydrofolate enters the reaction stoichiometrically (1 molof MTHF/1 mol of methionine formed), S-adenosylmethionine is convertedsubstoichiometrically as described in the literature. Cobalamin, on theother hand, is catalytically involved in the conversion. Further detailsof the metH protein are known to the skilled worker. (Banerjee R. V.,Matthews R. G., FASEB J., 4:1450-1459, 1990, Ludwig M L., Matthews R G.,Annual Review of Biochemistry. 66:269-313,1997, Drennan C L., Matthews RG., Ludwig M L., Current Opinion in Structural Biology. 4:919-29, 1994).The skilled worker distinguishes the activity of the cobalamin-dependent5-methyltetrahydrofolate homocysteine S-methyltransferase from that ofthe cobalamin-independent 5-methyltetrahydropteroyltriglutamatehomocysteine S-methyltransferase (EC 2.1.1.14), also known as metE. Theskilled worker can detect the enzymic activity of metH using enzymeassays, protocols for which may be: Jarrett J T., Goulding C W., FluhrK., Huang S., Matthews R G., Methods in Enzymology. 281:196-213, 1997.

Within the scope of the present invention, the term “sulfur-containingfine chemical” includes any chemical compound which contains at leastone covalently bound sulfur atom and is accessible by a fermentationmethod of the invention. Nonlimiting examples thereof are methionine,homocysteine, S-adenosylmethionine, in particular methionine andS-adenosylmethionine.

Within the scope of the present invention, the terms “L-methionine”,“methionine”, homocysteine and S-adenosylmethionine also include thecorresponding salts such as, for example, methionine hydrochloride ormethionine sulfate.

“Polynucleotides” in general refers to polyribonucleotides (RNA) andpolydeoxyribonucleotides (DNA) which may be unmodified RNA and DNArespectively, or modified RNA and DNA, respectively.

According to the invention, “polypeptides” means peptides or proteinswhich contain two or more amino acids linked via peptide bonds.

The term “metabolic metabolite” refers to chemical compounds which occurin the metabolism of organisms as intermediates or else final productsand which, apart from their property as chemical building blocks, mayalso have a modulating effect on enzymes and on their catalyticactivity. It is known from the literature that such metabolicmetabolites may act on the activity of enzymes in both an inhibiting anda stimulating manner (Biochemistry, Stryer, Lubert, 1995 W. H. Freeman &Company, New York, N.Y.). The possibility of producing in organismsenzymes in which the influence of metabolic metabolites has beenmodified by measures such as mutation of the genomic DNA by UVradiation, ionizing radiation or mutagenic substances and subsequentselection for particular phenotypes has also been described in theliterature (Sahm H., Eggeling L., de Graaf A A., Biological Chemistry381(9-10):899-910,2000; Eikmanns B J., Eggeling L., Sahm H., Antonie vanLeeuwenhoek., 64:145-63, 1993-94). These altered properties may also beachieved by specific measurements. The skilled worker knows how tomodify in enzyme genes specifically particular nucleotides of the DNAcoding for the protein in such a way that the protein resulting from theexpressed DNA sequence has certain new properties, for example that themodulating effect of metabolic metabolites on the unmodified protein haschanged.

The activity of enzymes may be influenced in such a way that thereaction rate is reduced or the affinity for the substrate is modifiedor the reaction rates are changed.

The terms “express” and “amplification” or “overexpression” describe inthe context of the invention the production of or increase inintracellular activity of one or more enzymes encoded by thecorresponding DNA in a microorganism. For this purpose, for example, itis possible to introduce a gene into an organism, to replace an existinggene by another gene, to increase the copy number of the gene or genes,to use a strong promoter or to use a gene which codes for acorresponding enzyme having a high activity, and these measures can becombined, where appropriate.

b) metH Proteins of the Invention

The invention likewise includes “functional equivalents” of thespecifically disclosed metH enzymes of organisms in the above list 1.

Within the scope of the present invention, “functional equivalents” oranalogs of the specifically disclosed polypeptides are polypeptidesdifferent therefrom, which furthermore have the desired biologicalactivity such as, for example, substrate specificity.

According to the invention, “functional equivalents” means in particularmutants which have in at least one of the abovementioned sequencepositions an amino acid other than the specifically mentioned aminoacid, but which have nevertheless one of the abovementioned biologicalactivities. “Functional equivalents” thus also include the mutantsobtainable by one or more amino acid additions, substitutions, deletionsand/or inversions, it being possible for said modifications to occur atany position in the sequence as long as they result in a mutant havingthe property profile of the invention. There is functional equivalencein particular also when the reaction patterns of mutant and unmodifiedpolypeptide match qualitatively, i.e. identical substrates are convertedwith different rates, for example.

“Functional equivalents” naturally also comprise polypeptides which areobtainable from other organisms, and naturally occurring variants. Forexample, homologous sequence regions can be found by sequencecomparison, and equivalent enzymes can be established following thespecific guidelines of the invention.

“Functional equivalents” likewise comprise fragments, preferablyindividual domains or sequence motifs, of the polypeptides of theinvention, which have the desired biological function, for example.

“Functional equivalents” are also fusion proteins which have one of theabovementioned polypeptide sequences or functional equivalents derivedtherefrom and at least one further heterologous sequence functionallydifferent therefrom in functional N— or C-terminal linkage (i.e. withnegligible functional impairment of the functions of the fusion proteinparts). Nonlimiting examples of such heterologous sequences are, forexample, signal peptides, enzymes, immunoglobulins, surface antigens,receptors or receptor ligands.

According to the invention, “functional equivalents” include homologs ofthe specifically disclosed proteins. These have, for example over theentire length, at least 30%, or about 40%, 50%, preferably at leastabout 60%, 65%, 70%, or 75%, in particular at least 85%, such as, forexample, 90%, 95% or 99%, homology to one of the specifically disclosedsequences, calculated by the algorithm of Pearson and Lipman, Proc.Natl. Acad., Sci. (USA) 85(8), 1988, 2444-2448. The degree of homologyreflects in particular the degree of identity between modified andunmodified sequence.

Homologs of the proteins or polypeptides of the invention can begenerated by mutagenesis, for example by point mutation or truncation ofthe protein. The term “homolog”, as used herein, also relates to avariant form of the protein, which acts as agonist or antagonist of theprotein activity.

Homologs of the proteins of the invention can be identified by screeningcombinatorial libraries of mutants such as, for example, truncationmutants. It is possible, for example, to generate a variegated libraryof protein variants by combinatory mutagenesis at the nucleic acidlevel, for example by enzymatic ligation of a mixture of syntheticoligonucleotides. There is a multiplicity of methods which can be usedfor preparing libraries of potential homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic gene can then be ligated into a suitable expression vector.The use of a degenerate set of genes makes it possible to provide wholesequences which encode the desired set of potential protein sequences inone mixture. Methods for synthesizing degenerate oligonucleotides areknown to the skilled worker (for example, Narang, S. A., (1983)Tetrahedron 39:3; Itakura et al., (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al., (1983) NucleicAcids Res. 11:477).

In addition, libraries of fragments of the protein codon can be used togenerate a variegated population of protein fragments for screening andfor subsequent selection of homologs of a protein of the invention. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double-stranded PCR fragment of a coding sequence with anuclease under conditions under which nicking occurs only about once permolecule, denaturing the double-stranded DNA, renaturing the DNA to formdouble-stranded DNA which may comprise sense/antisense pairs of variousnicked products, removing single-stranded sections from newly formedduplexes by treatment with S1 nuclease and ligating the resultingfragment library into an expression vector. It is possible by thismethod to devise an expression library which encodes N-terminal,C-terminal and internal fragments of the protein of the invention, whichhas different sizes.

Several techniques are known in the prior art for screening geneproducts from combinatorial libraries which have been produced by pointmutations or truncation and for screening cDNA libraries for geneproducts with a selected property. These techniques can be adapted torapid screening of gene libraries which have been generated bycombinatorial mutagenesis of homologs of the invention. The mostfrequently used techniques for screening large gene libraries undergoinghigh-throughput analysis comprise the cloning of the gene library intoreplicable expression vectors, transformation of suitable cells with theresulting vector library and expression of the combinatorial genes underconditions under which detection of the desired activity facilitatesisolation of the vector encoding the gene whose product has beendetected. Recursive ensemble mutagenesis (REM), a technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening tests in order to identifyhomologs (Arkin und Yourvan (1992) PNAS 89:7811-7815; Delgrave et al.(1993) Protein Engineering 6(3):327-331

c) Polynucleotides of the Invention

The invention also relates to nucleic acid sequences (single- anddouble-stranded DNA and RNA sequences such as, for example cDNA andmRNA) coding for one of the above metH enzymes and the functionalequivalents thereof which are obtainable, for example, also by use ofartificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules which codefor polypeptides or proteins of the invention or for biologically activesections thereof, and to nucleic acid fragments which can be used, forexample, for use as hybridization probes or primers for identifying oramplifying coding nucleic acids of the invention.

Moreover, the nucleic acid molecules of the invention may containuntranslated sequences from the 3′ and/or 5′ ends of the coding regionof the gene.

An “isolated” nucleic acid molecule is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acidand may moreover be essentially free of other cellular material orculture medium if it is prepared by recombinant techniques, or free ofchemical precursors or other chemicals if it is chemically synthesized.

The invention furthermore comprises the nucleic acid moleculescomplementary to the specifically described nucleotide sequences or asection thereof.

The nucleotide sequences of the invention make it possible to generateprobes and primers which can be used for identifying and/or cloninghomologous sequences in other cell types and organisms. Such probes andprimers usually complete a nucleotide sequence region which hybridizesunder stringent conditions to at least about 12, preferably at leastabout 25, such as, for example 40, 50 or 75, consecutive nucleotides ofa sense strand of a nucleic acid sequence of the invention or of acorresponding antisense strand.

Further nucleic acid sequences of the invention are derived from SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49 or 51 and differ therefrom through addition,substitution, insertion or deletion of one or more nucleotides, butstill code for polypeptides having the desired profile of properties.These may be polynucleotides which are identical to above sequences, forexample over the entire length, in at least about 50%, 55%, 60%, 65%,70%, 80% or 90%, preferably in at least about 95%, 96%, 97%, 98% or 99%,Of the sequence positions.

The invention also includes those nucleic acid sequences which comprise“silent” mutations or are modified, by comparison with a specificallymentioned sequence, in accordance with the codon usage of a specificsource or host organism, as well as naturally occurring variants suchas, for example, splice variants or allelic variants. The inventionlikewise relates to sequences which are obtainable by conservativenucleotide substitutions (i.e. the relevant amino acid is replaced by anamino acid of the same charge, size, polarity and/or solubility).

The invention also relates to molecules derived from specificallydisclosed nucleic acids through sequence polymorphisms. These geneticpolymorphisms may exist because of the natural variation betweenindividuals within a population. These natural variations usually resultin a variance of from 1 to 5% in the nucleotide sequence of a gene.

The invention furthermore also comprises nucleic acid sequences whichhybridize with or are complementary to the abovementioned codingsequences. These polynucleotides can be found on screening of genomic orcDNA libraries, and where appropriate, be amplified therefrom by meansof PCR using suitable primers, and then, for example, be isolated withsuitable probes. Another possibility is to transform suitablemicroorganisms with polynucleotides or vectors of the invention,multiply the microorganisms and thus the polynucleotides, and thenisolate them. An additional possibility is to synthesize polynucleotidesof the invention by chemical routes.

The property of being able to “hybridize” to polynucleotides means theability of a polynucleotide or oligonucleotide to bind under stringentconditions to an almost complementary sequence, while there are nounspecific bindings between noncomplementary partners under theseconditions. For this purpose, the sequences should be 70-100%,preferably 90-100%, complementary. The property of complementarysequences being able to bind specifically to one another is made use of,for example, in the Northern or Southern blot technique or in PCR orRT-PCR in the case of primer binding. Oligonucleotides with a length of30 base pairs or more are usually employed for this purpose. Stringentconditions means, for example, in the Northern blot technique the use ofa washing solution at 50-70° C., preferably 60-65° C., for example0.1×SSC buffer with 0.1% SDS (20×SSC; 3M NaCl, 0.3M Na citrate, pH 7.0)for eluting nonspecifically hybridized cDNA probes or oligonucleotides.In this case, as mentioned above, only nucleic acids with a high degreeof complementarity remain bound to one another. The setting up ofstringent conditions is known to the skilled worker and is described,for example, in Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. beschrieben.

c) Isolation of the Coding metH Gene

The metH genes coding for the enzyme methionine synthase (EC 2.1.1.13)can be isolated from the organisms of the above list I in a manner knownper se.

In order to isolate the metH genes or else other genes of the organismsof the above list I first a gene library of this organism is generatedin Escherichia coli (E. coli). The generation of gene libraries isdescribed in detail in generally known textbooks and manuals. Exampleswhich may be mentioned are the textbook by Winnacker: Gene und Klone,Eine Einführung in die Gentechnologie (Verlag Chemie, Weinheim, Germany,1990), and the manual by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A verywell-known gene library is that of E. coli K-12 strain W3110, which wasgenerated in λ vectors by Kohara et al. (Cell50, 495-508 (198).

In order to produce a gene library from organisms of list I in E. coli,cosmids such as the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84: 2160-2164), orelse plasmids such as pBR322 (BoliVal; Life Sciences, 25, 807-818(1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can be used.Suitable hosts are in particular those E. coli strains which arerestriction and recombination defective. An example of this is thestrain DH5αmcr which has been described by Grant et al. (Proceedings ofthe National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNAfragments cloned with the aid of cosmids may then in turn be subclonedinto common vectors suitable for sequencing and subsequently besequenced, as described, for example, in Sanger et al. (proceedings ofthe National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

The DNA sequences obtained can then be studied using known algorithms orsequence analysis programs such as, for example, those by Staden(Nucleic Acids Research 14, 217-232(1986)), by Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program by Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

The metH-encoding DNA sequences from organisms according to the abovelist I were found. In particular, DNA sequences according to SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49 and 51 were found. Furthermore, the aminoacid sequences of the corresponding proteins were derived from said DNAsequences present, using the above-described methods. SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50 and 52 depict the resulting amino acid sequences of themetH gene products.

Coding DNA sequences which result from the sequences according to SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49 and 51 due to the degeneracy of the geneticcode are likewise subject of the invention. In the same way, theinvention relates to DNA sequences which hybridize with said sequencesor parts of sequences derived therefrom.

Instructions for identifying DNA sequences by means of hybridization canbe found by the skilled worker, inter alia, in the manual “The DIGSystem Users Guide für Filter Hybridization” from Boehringer MannheimGmbH (Mannheim, Germany, 1993) and in Liebi et al. (InternationalJournal of Systematic Bacteriology (1991) 41: 255-260). Instructions foramplifying DNA sequences with the aid of the polymerase chain reaction(PCR) can be found by the skilled worker, inter alia, in the manual byGait: Oligonucleotide synthesis: A Practical Approach (IRL Press,Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum AkademischerVerlag, Heidelberg, Germany, 1994).

It is furthermore known that changes at the N— and/or C-terminus of aprotein do not substantially impair its function or may even stabilizesaid function. Information on this can be found by the skilled worker,inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757(1987)), in O'Regan et al. (Gene 77: 237-251 (1989), in Sahin-Toth etal. (Protein Sciences 3: 240-247 (1994)), in Hochuli et al.(Biotechnology 6: 1321-1325 (1988)) and in known textbooks of geneticsand molecular biology.

Amino acid sequences which result accordingly from SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50 and 52 are likewise part of the invention.

d) Host Cells Used According to the Invention

The invention further relates to microorganisms serving as host cells,in particular coryneform bacteria, which contain a vector, in particulara shuttle vector or plasmid vector, carrying at least one metH gene asdefined by the invention or in which a metH gene of the invention isexpressed or amplified.

These microorganisms can produce sulfur-containing fine chemicals, inparticular L-methionine, from glucose, sucrose, lactose, fructose,maltose, molasses, starch, cellulose or from glycerol and ethanol. Saidmicroorganisms are preferably coryneform bacteria, in particular of thegenus Corynebacterium. Of the genus Corynebacterium, mention must bemade in particular of the species Corynebacterium glutamicum which isknown in the art for its ability to produce L-amino acids.

Examples of suitable strains of coryneform bacteria, which may bementioned, are those of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum (C. glutamicum), such as

Corynebacterium glutamicum ATCC 13032,

Corynebacterium acetoglutamicum ATCC 15806,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium thermoaminogenes FERM BP-1539,

Corynebacterium melassecola ATCC 17965

or

of the genus Brevibacterium, such as

Brevibacterium flavum ATCC 14067

Brevibacterium lactofermentum ATCC 13869 and

Brevibacterium divaricatum ATCC 14020;

Or strains derived therefrom such as

Corynebacterium glutamicum KFCC10065

Corynebacterium glutamicum ATCC21608

which likewise produce the desired fine chemical or the precursor(s)thereof.

The abbreviation KFCC means the Korean Federation of Culture Collection,the abbreviation ATCC means the American Type Strain Culture Collection,and the abbreviation FERM means the collection of the National Instituteof Bioscience and Human Technology, Agency of Industrial Science andTechnology, Japan.

e) Carrying Out the Fermentation of the Invention

According to the invention, it was found that coryneform bacteria, afteroverexpression of a metH gene from organisms of the list I, producesulfur-containing fine chemicals, in particular L-methionine, in anadvantageous manner.

To achieve overexpression, the skilled worker can take differentmeasures individually or in combination. Thus it is possible to increasethe copy number of the appropriate genes or to mutate the promoter andregulatory region or the ribosomal binding site which is locatedupstream of the structural gene. Expression cassettes which areincorporated upstream of the structural gene act in the same way.Inducible promoters make it additionally possible to increase expressionduring the course of the fermentative L-methionine production.Expression is likewise improved by measures which extend the life spanof the mRNA. Furthermore, the enzymic activity is likewise enhanced bypreventing degradation of the enzyme protein. The genes or geneconstructs may be either present in plasmids with varying copy number orintegrated and amplified in the chromosome. A further possiblealternative is to achieve overexpression of the relevant genes, bychanging the media composition and management of the culture.

Instructions for this can be found by the skilled worker, inter alia, inMartin et al. (Biontechnology 5, 137-146 (1987)), in Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in theEuropean patent 0472869, in U.S. Pat. No. 4,601,893, in Schwarzer andPühler (Biotechnology 9, 84-87 (1991), in Remscheid et al. (Applied andEnvironmental Microbiology 60, 126-132 (1994), in LaBarre et al.(Journal of Bacteriology 175, 1001-1007 (1993)), in the patentapplication WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)),in the Japanese published specification JP-A-10-229891, in Jensen undHammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60: 512-538 (1996) and in knowntextbooks of genetics and molecular biology.

The invention therefore also relates to expression constructs comprisinga nucleic acid sequence coding for a polypeptide of the invention underthe genetic control of regulatory nucleic acid sequences; and to vectorscomprising at least one of said expression constructs. Such constructsof the invention preferably include a promoter 5′ upstream of theparticular coding sequence and a terminator sequence 3′ downstream andalso, where appropriate, further regulatory elements, in each caseoperatively linked to the coding sequence. An “operative linkage” meansthe sequential arrangement of promoter, coding sequence, terminator and,where appropriate, further regulatory elements such that each of theregulatory elements can properly carry out its function in theexpression of the coding sequence. Examples of operatively linkablesequences are activating sequences and enhancers and the like. Furtherregulatory elements include selectable markers, amplification signals,origins of replication and the like. Suitable regulatory sequences aredescribed, for example in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).

In addition to the artificial regulatory sequences, the naturalregulatory sequence may still be present upstream of the actualstructural gene. Genetic modification can, where appropriate, switch offthis natural regulation and increase or decrease expression of thegenes. However, the gene construct may also have a simpler design, i.e.no additional regulatory signals are inserted upstream of the structuralgene and the natural promoter with its regulation is not removed.Instead, the natural regulatory sequence is mutated such that regulationno longer takes place and gene expression is increased or reduced. Thegene construct may contain one or more copies of the nucleic acidsequences.

Examples of useful promoters are: ddh, amy, lysC, dapA, lysA fromCorynebacterium glutamicum promoters, but also Gram-positive promotersSPO2, as are described in Bacillus Subtilis and Its Closest Relatives,Sonenshein, Abraham L., Hoch, James A., Losick, Richard; ASM Press,District of Columbia, Washington and Patek M. Eikmanns B J., Patek J.,Sahm H., Microbiology. 142 1297-309, 1996 or else the cos, tac, trp,tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6,lambda-PR and lambda-PL promoters which are advantageously applied inGram-negative bacteria. Preference is also give to using induciblepromoters such as, for example light- and, in particular,temperature-inducible promoters such as the P_(r)P_(i) promoter. It isin principle possible to use all natural promoters with their regulatorysequences. In addition, it is also possible to use advantageouslysynthetic promoters.

The regulatory sequences mentioned are intended to make specificexpression of the nucleic acid sequences possible. Depending on the hostorganism, this may mean, for example, that the gene is expressed oroverexpressed only after induction, or that it is immediately expressedand/or overexpressed.

In this connection, the regulatory sequences and factors may preferablyhave a beneficial effect on, and thus increase or decrease, expression.Thus, it is possible and advantageous to enhance the regulatory elementsat the transcriptional level by using strong transcription signals suchas promoters and/or enhancers. However, it is also possible besides thisto enhance translation by, for example, improving the stability of themRNA.

An expression cassette is prepared by fusing a suitable promoter, asuitable Shine-Dalgamo sequence, to a metH nucleotide sequence and asuitable termination signal. For this purpose, common recombination andcloning techniques are used, such as those described, for example, inCurrent Protocols in Molecular Biology, 1993, John Wiley & Sons,Incorporated, New York, N.Y., PCR Methods, Gelfand, David H., Innis,Michael A., Sninsky, John J., 1999, Academic Press, Incorporated,Calif., San Diego, PCR Cloning Protocols, Methods in Molecular BiologySer., Vol. 192, 2nd ed., Humana Press, N.J., Totowa. T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J.Silhavy, M. L. Berman und L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and inAusubel, F. M. et al., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley Interscience (1987).

The recombinant nucleic acid construct or gene construct is expressed ina suitable host organism by inserting it advantageously into ahost-specific vector which makes optimal expression of the genes in thehost possible. Vectors are well known to the skilled worker and can befound, for example, in “Cloning Vectors” (Pouwels P. H. et al., Hrsg,Elsevier, Amsterdam-New York-Oxford, 1985). The term “vectors” means,apart from plasmids, also all other vectors known to the skilled worker,such as, for example, phages, transposons, IS elements, plasmids,cosmids and linear or circular DNA. These vectors can replicateautonomously in the host organism or are replicated chromosomally. MetHgenes of the invention were amplified by overexpressing them by way ofexample with the aid of episomal plasmids. Suitable plasmids are thosewhich are replicated in coryneform bacteria. Numerous known plasmidvectors such as, for example, pZ1 (Menkel et al., Applied andEnvironmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al.,Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74(1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Otherplasmid vectors such as, for example, pCLiK5MCS, or those based on pCG4(U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMSMicrobiology Letters 66, 119-124 (1990)) or pAG1 (U.S. Pat. No.5,158,891) may be used in the same way.

Suitable plasmid vectors are furthermore also those with the aid ofwhich it is possible to apply the method of gene amplification byintegration into the chromosome, as has been described, for example, byRemscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for the duplication and amplification of the hom-thrB operon. Inthis method, the complete gene is cloned into a plasmid vector which canreplicate in a host (typically E. coli) but not in C. glutamicum.Suitable vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), Bernard et al., Journal of Molecular Biology, 234: 534-541(1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al.,1986, Gene 41: 337-342). The plasmidvector containing the gene to be amplified is then transferred into thedesired C. glutamicum strain via transformation. Methods fortransformation are described, for example, in Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Biotechnology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)).

The activity of enzymes can be influenced by mutations in thecorresponding genes in such a way that the rate of the enzymic reactionis partly or completely reduced. Examples of such mutations are known tothe skilled worker (Motoyama H., Yano H., Terasaki Y., Anazawa H.,Applied & Environmental Microbiology. 67:3064-70,2001, Eikmanns B J.,Eggeling L., Sahm H., Antonie van Leeuwenhoek. 64:145-63, 1993-94.)

Additionally, it may be advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, toamplify, in addition to expression and amplification of a metH gene ofthe invention, one or more enzymes of the methionine biosyntheticpathway or of a biosynthetic or other metabolic pathway associatedtherewith (i.e. in functional connection therewith), such as of thecysteine, lysine or threonine metabolic pathway, such as in particularof aspartate-semialdehyde synthesis, of glycolysis, of anaplerosis, ofthe pentose phosphate metabolism, the citrate acid cycle or the aminoacid export.

Thus, one or more of the following genes can be amplified to producesulfur-containing fine chemicals, in particular L-methionine, (i.e. forexample, be present with a higher copy number or encode an enzyme withhigher activity or specificity):

-   -   the aspartate kinase-encoding gene lysC (EP 1 108 790 A2;        DNA-SEQ NO.281),    -   the aspartate-semialdehyde dehydrogenase-encoding gene asd (EP 1        108 790 A2; DNA-SEQ NO. 282),    -   the glyceraldehyde-3-phosphate dehydrogenase-encoding gene gap        (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),    -   the 3-phosphoglycerate kinase-encoding gene pgk (Eikmanns        (1992), Journal of Bacteriology 174: 6076-6086),    -   the pyruvate carboxylase-encoding gene pyc (Eikmanns (1992),        Journal of Bacteriology 174: 6076-6086),    -   the triose phosphate isomerase-encoding gene tpi (Eikmanns        (1992), Journal of Bacteriology 174: 6076-6086),    -   the homoserine O-acetyltransferase-encoding gene metA (EP 1 108        790 A2; DNA-SEQ NO. 725),    -   the cystathionine gamma-synthase-encoding gene metB (EP 1 108        790 A2; DNA-SEQ NO. 3491),    -   the cystathionine gamma-lyase-encoding gene metC (EP 1 108 790        A2; DNA-SEQ NO. 3061),    -   the serine hydroxymethyltransferase-encoding gene glyA (EP 1 108        790 A2; DNA-SEQ NO. 1110),    -   the O-acetylhomoserine sulfhydrylase-encoding gene metY (EP 1        108 790 A2; DNA-SEQ NO. 726),    -   the methylene tetrahydrofolate reductase-encoding gene metF (EP        1 108 790 A2; DNA-SEQ NO. 2379),    -   the phosphoserine aminotransferase-encoding gene serC (EP 1 108        790 A2; DNA-SEQ NO. 928),    -   a phosphoserine phosphatase-encoding gene serB (EP 1 108 790 A2;        DNA-SEQ NO. 334, DNA-SEQ NO. 467, DNA-SEQ NO. 2767),    -   the serine acetyl transferase-encoding gene cysE (EP 1 108 790        A2; DNA-SEQ NO. 2818),    -   the homoserine dehydrogenase-encoding gene hom (EP 1 108 790 A2;        DNA-SEQ NO. 1306)

Thus, it may be advantageous for the production of sulfur-containingfine chemicals, in particular L-methionine, in coryneform bacteria tomutate, at the same time, at least one of the genes below, in particularso that the activity of the corresponding proteins, compared to that ofunmutated proteins, is influenced by a metabolic metabolite to a lesserextent or not at all:

-   -   the aspartate kinase-encoding gene lysC (EP 1 108 790 A2;        DNA-SEQ NO. 281),    -   the pyruvate carboxylase-encoding gene pyc (Eikmanns (1992),        Journal of Bacteriology 174: 6076-6086),    -   the homoserine O-acetyltransferase-encoding gene metA (EP 1 108        790 A2; DNA-SEQ NO. 725),    -   the cystathionine gamma-synthase-encoding gene metB (EP 1 108        790 A2; DNA-SEQ NO. 3491),    -   the cystathionine gamma-lyase-encoding gene metC (EP 1 108 790        A2; DNA-SEQ NO. 3061),    -   the serine hydroxymethyltransferase-encoding gene glyA (EP 1 108        790 A2; DNA-SEQ NO. 1110),    -   the 0-acetylhomoserine sulfhydrylase-encoding gene metY (EP 1        108 790 A2; DNA-SEQ NO. 726),    -   the methylene tetrahydrofolate reductase-encoding gene metF (EP        1 108 790 A2; DNA-SEQ NO. 2379),    -   the phosphoserine aminotransferase-encoding gene serC (EP 1 108        790 A2; DNA-SEQ NO. 928),    -   a phosphoserine phosphatase-encoding gene serB (EP 1 108 790 A2;        DNA-SEQ NO. 334, DNA-SEQ NO. 467, DNA-SEQ NO. 2767),    -   the serine acetyl transferase-encoding gene cysE (EP 1 108 790        A2; DNA-SEQ NO. 2818),    -   the homoserine dehydrogenase-encoding gene hom (EP 1 108 790 A2;        DNA-SEQ NO. 1306)

It may be furthermore advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, inaddition to expression and amplification of one of the metH genes of theinvention, to attenuate one or more of the following genes, inparticular to reduce expression thereof, or to switch them off:

-   -   the homoserine kinase-encoding gene thrB (EP 1 108 790 A2;        DNA-SEQ NO. 3453),    -   the threonine dehydratase-encoding gene ilvA (EP 1 108 790 A2;        DNA-SEQ NO. 2328),    -   the threonine synthase-encoding gene thrC (EP 1 108 790 A2;        DNA-SEQ NO. 3486),    -   the meso-diaminopimelate D-dehydrogenase-encoding gene ddh (EP 1        108 790 A2; DNA-SEQ NO. 3494),    -   the phosphoenolpyruvate carboxykinase-encoding gene pck (EP 1        108 790 A2; DNA-SEQ NO. 3157),    -   the glucose-6-phosphate 6-isomerase-encoding gene pgi (EP 1 108        790 A2; DNA-SEQ NO. 950),    -   the pyruvate oxidase-encoding gene poxB (EP 1 108 790 A2;        DNA-SEQ NO. 2873),    -   the dihydrodipicolinate synthase-encoding gene dapA (EP 1 108        790 A2; DNA-SEQ NO. 3476),    -   the dihydrodipicolinate reductase-encoding gene dapB (EP 1 108        790 A2; DNA-SEQ NO. 3477)    -   the diaminopicolinate decarboxylase-encoding gene lysA (EP 1 108        790 A2; DNA-SEQ NO. 3451)

It may be furthermore advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, inaddition to expression and amplification of one of the metH genes of theinvention in coryneform bacteria, to mutate, at the same time, at leastone of the following genes in such a way that the enzymic activity ofthe corresponding protein is partly or completely reduced:

-   -   the homoserine kinase-encoding gene thrB (EP 1 108 790 A2;        DNA-SEQ NO. 3453),    -   the threonine dehydratase-encoding gene ilvA (EP 1 108 790 A2;        DNA-SEQ NO. 2328),    -   the threonine synthase-encoding gene thrC (EP 1 108 790 A2;        DNA-SEQ NO. 3486),    -   the meso-diaminopimelate D-dehydrogenase-encoding gene ddh (EP 1        108 790 A2; DNA-SEQ NO. 3494),    -   the phosphoenolpyruvate carboxykinase-encoding gene pck (EP 1        108 790 A2; DNA-SEQ NO. 3157),    -   the glucose-6-phosphate 6-isomerase-encoding gene pgi (EP 1 108        790 A2; DNA-SEQ NO. 950),    -   the pyruvate oxidase-encoding gene poxB (EP 1 108 790 A2;        DNA-SEQ NO. 2873),    -   the dihydrodipicolinate synthase-encoding gene dapA (EP 1 108        790 A2; DNA-SEQ NO. 3476),    -   the dihydrodipicolinate reductase-encoding gene dapB (EP 1 108        790 A2; DNA-SEQ NO. 3477)    -   the diaminopicolinate decarboxylase-encoding gene lysA (EP 1 108        790 A2; DNA-SEQ NO. 3451)

It may be furthermore advantageous for the production ofsulfur-containing fine chemicals, in particular L-methionine, apart fromexpression and amplification of a metH gene of the invention, toeliminate unwanted secondary reactions which for example reduce theyield of fine chemicals (Nakayama: “Breeding of Amino Acid ProducingMicroorganisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention may be culturedcontinuously or batchwise or in a fed batch or repeated fed batchprocess to produce sulfur-containing fine chemicals, in particularL-methionine. An overview of known cultivation methods can be found inthe textbook by Chmiel (Bioprozeβtechnik 1. Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must satisfy the demands of the particularstrains in a suitable manner. The textbook “Manual of Methods fürGeneral Bacteriology” by the American Society for Bacteriology(Washington D.C., USA, 1981) contains descriptions of culture media forvarious microorganisms.

Said media which can be used according to the invention usually compriseone or more carbon sources, nitrogen sources, inorganic salts, vitaminsand/or trace elements.

Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch and cellulose. Sugars may also beadded to the media via complex compounds such as molasses or otherbyproducts of sugar refining. It may also be advantageous to addmixtures of different carbon sources. Other possible carbon sources areoils and fats such as, for example, soybean oil, sunflower oil, peanutoil and coconut fat, fatty acids such as, for example, palmitic acid,stearic acid and linoleic acid, alcohols such as, for example, glycerol,methanol and ethanol and organic acids such as, for example acetic acidand lactic acid.

Nitrogen sources are usually organic or inorganic hydrogen compounds ormaterials containing said compounds. Examples of nitrogen sourcesinclude ammonia gas or ammonium salts such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,nitrates, urea, amino acids and complex nitrogen sources such ascornsteep liquor, soybean flour, soybean protein, yeast extract, meatextract and others. The nitrogen sources may be used singly or asmixture.

Inorganic salt compounds which may be included in the media comprise thechloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used assources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention usually alsocontain other growth factors such as vitamins or growth promoters, whichinclude, for example, biotin, riboflavin, thiamine, folic acid,nicotinic acid, panthothenate and pyridoxine. Growth factors and saltsare frequently derived from complex media components such as yeastextract, molasses, cornsteep liquor and the like. It is moreoverpossible to add suitable precursors to the culture medium. The exactcomposition of the media heavily depends on the particular experimentand is decided upon individually for each specific case. Information onthe optimization of media can be found in the textbook “AppliedMicrobiol. Physiology, A Practical Approach” (Editors. P. M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growthmedia can also be obtained from commercial suppliers, for exampleStandard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by sterile filtration. The components may be sterilizedeither together or, if required, separately. All media components may bepresent at the start of the cultivation or added continuously orbatchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° C., and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, air intothe culture. The temperature of the culture is normally 20° C. to 45° C.The culture is continued until formation of the desired product is at amaximum. This aim is normally achieved within 10 to 160 hours.

The fermentation broths obtained in this way, in particular thosecontaining L-methionine, usually contain a dry biomass of from 7.5 to25% by weight.

An additional advantage is to carry out the fermentation under sugarlimitation, at least at the end, but in particular over at least 30% ofthe fermentation period. This means that during this time theconcentration of utilizable sugar in the fermentation medium ismaintained at or reduced to ≧0 to 3 g/l.

The fermentation broth is then processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth.

Subsequently, the fermentation broth may be thickened or concentratedusing known methods such as, for example, with the aid of a rotaryevaporator, thin film evaporator, falling film evaporator, by reverseosmosis, or by nanofiltration. This concentrated fermentation broth canthen be worked up by freeze drying, spray drying, spray granulation orby other methods.

However, it is also possible to further purify the sulfur-containingfine chemicals, in particular L-methionine. To this end, theproduct-containing broth, after removing the biomass, is subjected to achromatography using a suitable resin, the desired product or thecontaminations being retained completely or partially on thechromatographic resin. These chromatographic steps can be repeated, ifnecessary, using the same or different chromatographic resin. Theskilled worker is familiar with the selection of suitablechromatographic resins and their most effective application. Thepurified product can be concentrated by filtration or ultrafiltrationand stored at a temperature at which the stability of the product isgreatest.

The identity and purity of the isolated compound(s) can be determined bytechniques of the art. These include high performance liquidchromatography (HPLC), spectroscopic methods, staining methods,thin-layer chromatography, NIRS, enzyme assay or microbiological assays.These analytic methods are summarized in: Patek et al. (1994) Appl.Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.Ulmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH:Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 andpp. 581-587; Michal, G., (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, Volume 17.

The following nonlimiting examples describe the invention in moredetail:

EXAMPLE 1 Construction of pCLiK5MCS

First, ampicillin resistance and origin of replication of the vectorpBR322 were amplified using the oligonucleotides p1.3 (SEQ ID NO:53) andp2.3 (SEQ ID NO:54) with the aid of the polymerase chain reaction (PCR).

p1.3 (SEQ ID NO:53) 5′-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3′ p2.3 (SEQ ID NO:54)5′-TCTAGACTCGAGCGGCCGCGGCCGGCCT TTAAATTGAAGACGAAAGGGCCTCG-3′

In addition to sequences complementary to pBR322, the oligonucleotidep1.3 (SEQ ID NO:53) contains in 5′-3′ direction the cleavage sites forthe restriction nucleases SmaI, BamHI, NheI and AscI and theoligonucleotide p2.3 (SEQ ID NO:54) contains in 5′-3′ direction thecleavage sites for the restriction endonucleases XbaI, XhoI, NotI andDraI. The PCR reaction was carried out according to a standard methodsuch as that by Innis et al. (PCR Protocols. A Guide to Methods andApplications, Academic Press (1990)) using PfuTurbo polymerase(Stratagene, La Jolla, USA). The DNA fragment obtained of approximately2.1 kb in size was purified using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. The blunt ends of the DNA fragment wereligated to one another using the rapid DNA ligation kit (RocheDiagnostics, Mannheim) according to the manufacturer's instructions andthe ligation mixture was transformed into competent E. coli XL-1Blue(Stratagene, La Jolla, USA) according to standard methods, as describedin Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold SpringHarbor, (1989)). Plasmid-carrying cells were selected for by plating outonto ampicillin (50 μg/ml)-containing LB agar (Lennox, 1955, Virology,1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK1.

Starting from plasmid pWLT1 (Liebl et al., 1992) as template for a PCRreaction, a kanamycin resistance cassette was amplified using theoligonucleotides neo1 (SEQ ID NO:55) and neo2 (SEQ ID NO:56).

neo1 (SEQ ID NO:55): 5′-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGGA-3′ neo2 (SEQ ID NO:56):5′-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3′

Apart from the sequences complementary to pWLT1, the oligonucleotideneo1 contains in 5′-3′ direction the cleavage sites for the restrictionendonucleases XbaI, SmaI, BamHI, NheI and the oligonucleotide neo2 (SEQID NO:56) contains in 5′-3′ direction the cleavage sites for therestriction endonucleases AscI and NheI. The PCR reaction was carriedout using PfuTurbo polymerase (Stratagene, La Jolla, USA) according to astandard method such as that of Innis et al. (PCR Protocols. A Guide toMethods and Applications, Academic Press (1990)). The DNA fragmentobtained was approximately 1.3 kb in size was purified using theGFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. The DNA fragmentwas cleaved with restriction endonucleases XbaI and AscI (New EnglandBiolabs, Beverly, USA) and, following that, again purified using theGFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. The vectorpCLiK1 was likewise cleaved with the restriction endonucleases XbaI andAscI and dephosphorylated using alkaline phosphatase (Roche Diagnostics,Mannheim) according to the manufacturer's instructions. Afterelectrophoresis in a 0.8% strength agarose gel, the linearized vector(approx. 2.1 kb) was isolated using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. This vector fragment was ligated with thecleaved PCR fragment with the aid of the rapid DNA ligation kit (RocheDiagnostics, Mannheim) according to the manufacturer's instructions andthe ligation mixture was transformed into competent E. coli XL-1Blue(Stratagene, La Jolla, USA) according to standard methods, as describedin Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold SpringHarbor, (1989)). Plasmid-carrying cells were selected for by plating outonto ampicillin (50 μg/ml)- and kanamycin (20 μg/ml)-containing LB agar(Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK2.

The vector pCLiK2 was cleaved with the restriction endonuclease DraI(New England Biolabs, Beverly, USA). After electrophoresis in 0.8%strength agarose gel, an approx. 2.3 kb vector fragment was isolatedusing the GFX™PCR, DNA and gel band purification kit (AmershamPharmacia, Freiburg) according to the manufacturer's instructions. Thisvector fragment was religated with the aid of the rapid DNA ligation kit(Roche Diagnostics, Mannheim) according to the manufacturer'sinstructions and the ligation mixture was transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA) according to standard methods,as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual,Cold Spring Harbor, (1989)). Plasmid-carrying cells were selected for byplating out onto kanamycin (20 μg/ml)-containing LB agar (Lennox, 1955,Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK3.

Starting from plasmid pWLQ2 (Liebl et al., 1992) as template for a PCRreaction, the origin of replication pHM1519 was amplified using theoligonucleotides cg1 (SEQ ID NO:57) and cg2 (SEQ ID NO:58).

cg1 (SEQ ID NO:57): 5′-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-3′ cg2 (SEQ IDNO:58): 5′-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3′

Apart from the sequences complementary to pWLQ2, the oligonucleotidescg1 (SEQ ID NO:57) and cg2 (SEQ ID NO:58) contain cleavage sites for therestriction endonuclease NotI. The PCR reaction was carried out usingPfuTurbo polymerase (Stratagene, La Jolla, USA) according to a standardmethod such as that of Innis et al. (PCR Protocols. A Guide to Methodsand Applications, Academic Press (1990)). The DNA fragment obtained wasapproximately 2.7 kb in size and was purified using the GFX™PCR, DNA andgel band purification kit (Amersham Pharmacia, Freiburg) according tothe manufacturer's instructions. The DNA fragment was cleaved withrestriction endonuclease NotI (New England Biolabs, Beverly, USA) and,following that, again purified using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. The vector pCLiK3 was likewise cleaved withthe restriction endonuclease NotI and dephosphorylated using alkalinephosphatase (Roche Diagnostics, Mannheim) according to themanufacturer's instructions. After electrophoresis in a 0.8% strengthagarose gel, the linearized vector (approx. 2.3 kb) was isolated usingthe GFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. This vectorfragment was ligated with the cleaved PCR fragment with the aid of therapid DNA ligation kit (Roche Diagnostics, Mannheim) according to themanufacturer's instructions and the ligation mixture was transformedinto competent E. coli XL-1Blue (Stratagene, La Jolla, USA) according tostandard methods, as described in Sambrook et al. (Molecular Cloning. ALaboratory Manual, Cold Spring Harbor, (1989)). Plasmid-carrying cellswere selected for by plating out onto kanamycin (20 μg/ml)-containing LBagar (Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK5.

PCLik5 was extended by a multiple cloning site (MCS) by combining thetwo synthetic essentially complementary oligonucleotides HS445 ((SEQ IDNO:59) and HS446 (SEQ ID NO:60)) which contain cleavage sites for therestriction endonucleases SwaI, XhoI, AatI, ApaI, Asp718, MluI, NdeI,SpeI, EcoRV, SalI, ClaI, BamHI, XbaI and SmaI to give a double-strandedDNA fragment by heating them together to 95° C. followed by slowcooling.

HS445 (SEQ ID NO:59): 5′-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATATGACTAGTTCGGACCTAGGGATATCGTCGACATCGATGCTCTTCTGCGTTAATTAACAATTGGGATCCTCTAGACCCGGGATTTAAAT-3′ HS446 (SEQ ID NO:60):5′-GATCATTTAAATCCCGGGTCTAGAGGATCCCAATTGTTAATTAACGCAGAAGAGCATCGATGTCGACGATATCCCTAGGTCCGAACTAGTCATATGACGCGTGGTACCGGGCCCGACGTCAGGCCTCTCGAGATTTAAAT-3′

The vector pCLiK5 was cleaved with the restriction endonucleases XhoIand BamHI (New England Biolabs, Beverly, USA) and dephosphorylated usingalkaline phosphatase (I (Roche Diagnostics, Mannheim)) according to themanufacturer's instructions. After electrophoresis in a 0.8% strengthagarose gel, the linearized vector (approx. 5.0 kb) was isolated usingthe GFX™PCR, DNA and gel band purification kit (Amersham Pharmacia,Freiburg) according to the manufacturer's instructions. This vectorfragment was ligated with the synthetic double-stranded DNA fragmentwith the aid of the rapid DNA ligation kit (Roche Diagnostics, Mannheim)according to the manufacturer's instructions and the ligation mixturewas transformed into competent E. coli XL-1Blue (Stratagene, La Jolla,USA) according to standard methods as described Sambrook et al.(Molecular Cloning. A Laboratory Manual, Cold Spring Harbor (1989)).Plasmid-carrying cells were selected for by plating out onto kanamycin(20 μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK5MCS.

Sequencing reactions were carried out according to Sanger et al. (1977)Proceedings of the National Academy of Sciences USA 74:5463-5467. Thesequencing reactions were fractionated and analyzed by means of ABIPrism 377 (PE Applied Biosystems, Weiterstadt).

The resultant plasmid pCLiK5MCS is listed as SEQ ID NO: 63.

EXAMPLE 2 Construction of pCLiK5MCS Integrativ sacB

Starting from the plasmid pK19mob (Schäfer et al., Gene 145,69-73(1994))as template for a PCR reaction, the Bacillus subtilis sacB gene (codingfor levan sucrase) was amplified using the oligonucleotides BK1732 andBK1733.

BK1732 (SEQ ID NO:61): 5′-GAGAGCGGCCGCCGATCCTTTTTAACCCATCAC-3′ BK1733(SEQ ID NO:62): 5′-AGGAGCGGCCGCCATCGGCATTTTCTTTTGCG-3′

Apart from the sequences complementary to pEK19mobsac, theoligonucleotides BK1732 and BK1733 contain cleavage sites for therestriction endonuclease NotI. The PCR reaction was carried out usingPfuTurbo polymerase (Stratagene, La Jolla, USA) using a standard methodlike that of Innis et al. (PCR Protocols. A Guide to Methods andApplications, Academic Press (1990)). The DNA fragment obtained ofapproximately 1.9 kb in size was purified using the GFX™PCR, DNA and gelband purification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions. The DNA fragment was cleaved with therestriction endonuclease NotI (New England Biolabs, Beverly, USA) and,following that, again purified using the GFX™PCR, DNA and gel bandpurification kit (Amersham Pharmacia, Freiburg) according to themanufacturer's instructions.

The vector pCLiK5MCS (prepared according to example 1) was likewisecleaved with the restriction endonuclease NotI and dephosphorylatedusing alkali phosphatase (I (Roche Diagnostics, Mannheim)) according tothe manufacturer's instructions. After electrophoresis in a 0.8%strength agarose gel, an approximately 2.4 kb in size vector fragmentwas isolated using the GFX™PCR, DNA and gel band purification kit(Amersham Pharmacia, Freiburg) according to the manufacturer'sinstructions. This vector fragment was ligated with the cleaved PCRfragment with the aid of the rapid DNA ligation kit (Roche Diagnostics,Mannheim) according to the manufacturer's instructions and the ligationmixture was transformed into competent E. coli XL-1Blue (Stratagene, LaJolla, USA) according to standard methods, as described in Sambrook etal. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,(1989)). Plasmid-carrying cells were selected for by plating out ontokanamycin (20 μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) according to the manufacturer'sinstructions and checked by restriction digests. The plasmid obtained inthis way is denoted pCLiK5MCS integrativ sacB.

Sequencing reactions were carried out according to Sanger et al. (1977)Proceedings of the National Academy of Sciences USA 74:5463-5467. Thesequencing reactions were fractionated and analyzed by means of ABIPrism 377 (PE Applied Biosystems, Weiterstadt).

The resultant plasmid pCLiK5MCS integrativ sacB is listed as SEQ ID NO:64.

It is possible to prepare in an analog manner further vectors which aresuitable for the inventive expression or overproduction of metH genes.

Examples 3 to 8 hereinbelow describe the step-wise construction of animproved methionine-producing strain referred to as LU1479 lysC 311ileET-16 pC Phsdh metH Sc.

EXAMPLE 3 Isolation of the LysC Gene from C. glutamicum Strain LU1479

An allelic exchange of the lysC wild-type gene encoding the enzymeaspartate kinase in C. glutamicum ATCC13032, hereinbelow referred to asLU1479, is intended to be carried out in the first step of stemconstruction. A nucleotide exchange is to be carried out in the LysCgene so that the amino acid Thr at position 311 is exchanged in theresulting protein for the amino acid Ile.

Starting from the LU1479 chromosomal DNA as template for a PCR reaction,amplification was performed with the oligonucleotide primers SEQ IDNO:65 and SEQ ID NO:66 lysC with the aid of the Pfu-Turbo PCR System(Stratagene USA), following the manufacturer's instructions. C.glutamicum ATCC 13032 chromosomal DNA was prepared following the methodof Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)Microbiology 140:1817-1828. The amplified fragment is flanked by an SalIrestriction cleavage at its 5′ end and by an MluI restriction cleavageat its 3′ end. Prior to cloning, the amplified fragment was digested bythese two restriction enzymes and purified using GFX™PCR, DNA and GelBand Purification Kit (Amersham Pharmacia, Freiburg).

SEQ ID NO:65 5′-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3′ SEQ ID NO:665′-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3′

The resulting polynucleotide was cloned into pCLIK5 MCS integrativ SacB(hereinbelow referred to as pCIS; SEQ ID NO:64 of Example 2) via theSalI and MluI reaction cleavages and transformed into E. coli XL-1blue.Selection for plasmid-bearing cells was achieved by plating on kanamycin(20μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190). Theplasmid was isolated, and the expected nucleotide sequence was verifiedby sequencing. Preparation of the plasmid DNA was carried out by methodsof, and using materials from, Quiagen. Sequencing reactions were carriedout as described by Sanger et al. (1977) Proceedings of the NationalAcademy of Sciences USA 74:5463-5467. The sequencing reactions wereseparated and evaluated by means of ABI Prism 377 (PE AppliedBiosystems, Weiterstadt). The resultant plasmid pCIS lysC is listed asSEQ ID NO:77.

Sequence SEQ ID NO:77 comprises the following essential part-regions:

Type of Position sequence Description  155-1420 CDS¹⁾ lysC 1974-2765 CDSkanamycin resistance 3032-3892 CDS replication origin/E. coli/(complement)²⁾ plasmid pMB ¹⁾coding sequence ²⁾on complementary strain

EXAMPLE 4 Mutagenesis of the C. glutamicum LysC Gene

The directed mutagenesis of the C. glutamicum lysC gene (Example 3) wascarried out using the QuickChange Kit (Stratagene/USA) following themanufacturer's instructions. The mutagenesis was carried out in plasmidpCIS lysC, SEQ ID NO:77. The following oligonucleotide primers weresynthesized with the aid of the Quickchange method (Stratagene) for theexchange of thr311 for 311 ile:

SEQ ID NO:67 5′-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3′ SEQ ID NO:685′-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3′

The use of these oligonucleotide primers in the Quickchange reactionbrings about an exchange of the nucleotide in position 932 (C beingreplaced by T) (cf. SEQ ID NO:75) in the lysC gene and to an amino acidsubstitution in position 311 (Thr→Ile) (cf. SEQ ID NO:76) in thecorresponding enzyme. The resulting amino acid exchange Thr311ile in thelysC gene was verified by sequencing following transformation into E.coli XL1-blue and plasmid preparation.

The plasmid was named pCIS lysC thr311ile and is listed as SEQ ID NO:78.

Sequence ID NO:78 comprises the following essential part-regions:

Type of Position sequence Description  155-1420 CDS¹⁾ lysC mutated1974-2765 CDS kanamycin resistance 3032-3892 CDS replication origin/E.coli/ (complement)²⁾ plasmid pMB ¹⁾coding sequence ²⁾on complementarystrain

Plasmid pCIS lysC thr311ile was transformed into C. glutamicum LU1470 bymeans of electroporation as described by Liebl, et al. (1989) FEMSMicrobiology Letters 53:299-303. Modifications of the protocol aredescribed in DE-A-10046870. The chromosomal arrangement of the lysClocus of individual transformants was verified by standard methods usingSouthern blotting and hybridization as described by Sambrook et al.(1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor. Thiswas done to ensure that the transformants are transformants which havethe transformed plasmid integrated at the lysC locus by homologousrecombination. Such colonies were grown overnight in media withoutantibiotic and the cells were then plated onto sucrose CM agar medium(10% sucrose) and incubated for 24 hours at 30° C.

Since the sacB gene, which is present in the vector pCIS lysC thr311ile,converts sucrose into a toxic product, only those colonies which havethe sacB gene deleted between the wild-type lysC gene and the mutatedgene lysC thr311ile by a second homologous recombination step arecapable of establishing growth. During the homologous recombinationstep, either the wild-type gene or the mutated gene may be deletedtogether with the sacB gene. If the sacB gene is removed together withthe wild-type gene, a mutated transformant results.

Colonies with established growth were picked and studied forkanamycin-sensitive penotypes. Clones with the delected SacB gene mustsimultaneously display kanamycin-sensitive growth behavior. SuchKan-sensitive clones were studied in a shake flask for their lysinproductivity (see Example 6). For comparison, the untreated strainLU1479 was grown. Clones whose lysine production exceeds that of thecontrol were selected, chromosomal DNA was obtained, and the matchingregion of the LysC gene was amplified by a PCR reaction and sequenced.Such a clone with the property of increased lysine synthesis andconfirmed mutation in lysC at position 932 was referred to as LU1479lysC 311ile).

EXAMPLE 5 Generation of Ethionine-Resistant C. glutamicum Strains

In the second step of stem construction, the resulting strain LU1479lysC 311ile (Example 4) was treated in order to induce ethionineresistance (Kase, H. Nakayama K. Agr. Biol. Chem. 39 153-106 1975L-methionine production by methionine analog-resistant mutants ofCorynebacterium glutamicum): an overnight culture in BHI medium (Difco)was washed in citrate buffer (50 mM pH 5.5) and treated for 20 minutesat 30° C. with N-methylnitrosoguanidine (10 mg/ml in 50 mM citrate pH5.5). After treatment with the chemical mutagenN-methylnitrosoguanidine, the cells were washed (citrate buffer 50 mM pH5.5) and plated onto a medium composed of the following components,based on 500 ml: 10 g (NH₄)₂SO₄, 0.5 g KH₂PO₄, 0.5 g K₂HPO₄, 0.125 gMgSO₄.7H₂O, 21 g MOPS, 50 mg CaCl₂, 15 mg proteocatechuate, 0.5 mgbiotin, 1 mg thiamine, 5 g/l D,L-ethionine (Sigma Chemicals Germany), pH7.0. In addition, the medium comprised 0.5 ml of a micronutrient saltsolution of: 10 g/l FeSO₄.7H₂O, 1 g/l MnSO_(4*)H₂O, 0.1 g/l ZnSO₄*7H₂O,0.02 g/l CuSO₄, 0.002 g/l NiCl₂*6H₂O. All the salts were dissolved in0.1 M HCl. The finished medium was filtered-sterilized, 40 ml of sterile50% glucose solution was added, liquid sterile agarwas added to a finalconcentration of 1.5% and the mixture was poured into culture dishes.

Cells which had undergone mutagenizing treatment were applied to platescontaining the above-described medium and incubated for 3-7 days at 30°C. Resulting clones were isolated, isolated at least once on theselection medium and then tested for their methionine productivity inmedium II in a shake flask (see Example 6).

EXAMPLE 6 Production of Methionine Using Strain LU1479 LysC 311ile ET-16

The strains generated in Example 5 were grown for 2 days at 30° C. on anagar plate containing CM medium.

CM agar:

10.0 g/l D-Glucose, 2.5 g/l NaCl, 2.0 g/l urea, 10.0 g/l Bacto-peptone(Difco), 5.0 g/l yeast extract (Difco), 5.0 g/l beef extract (Difco),22.0 g/l agar (Difco), autoclaved (20 min., 121° C.)

The cells were subsequently scraped from the plate and resuspended insaline. For the main culture, 10 ml of medium II and 0.5 g of autoclavedCaCO₃ (Riedel de Haen) in a 100 ml Erlenmeyer flask were inoculated withthe cell suspension to an OD600 nm of 1.5 and incubated for 72 hours at30° C. on an orbital shaker at 200 rpm.

Medium II:

40 g/l sucrose 60 g/l molasses (based on 100% sugar content) 10 g/l(NH₄)₂SO₄ 0.4 g/l MgSO₄*7H₂O 0.6 g/l KH₂PO₄ 0.3 mg/l thiamine*HCl 1 mg/lbiotin (from a 1 mg/ml filter-sterilized stock solution which had beenbrought to pH 8.0 with NH₄OH 2 mg/l FeSO₄ 2 mg/l MnSO₄brought to pH 7.8 with NH₄OH and autoclaved (121° C., 20 min). Inaddition, vitamin B12 (hydroxycobalamine, Sigma Chemicals) from a stocksolution (200 μg/ml, filter-sterilized) is added to a finalconcentration of 100 μg/l.

Methionine formed, and other amino acids in the culture broth, was withthe aid of the amino acid determination method from Agilent on anAgilent 1100 Series LC System HPLC. Derivatization withortho-phthalaldehyde before the column separation allowed thequantification of the amino acids formed. The amino acid mixture wasseparated on a Hypersil AA column (Agilent).

Clones whose methionine productivity was at least twice as high as thatof the original strain LU 1479 lysC 311ile were isolated. One such aclone was employed in the subsequent experiments and was named LU 1479lysC 311ile ET-16.

EXAMPLE 7 Cloning metH from Streptomyces coelicolor and Cloning intoPlasmid pCPhsdh metH Sc

a) Chromosomal DNA was isolated from Streptomyces coelicolor strain ATCCBAA-471 (from the American Type Strain Culture Collection, (ATCC)Atlanta, USA, available under the catalog number BAA-471 D). C.glutamicum ATCC 13032 chromosomal DNA was prepared by the method ofTauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)Microbiology 140:1817-1828.

A DNA fragment of approx. 180 base pairs in length was amplified fromthe noncoding 5′ region (promoter region) of homoserine dehydrogenase(HsDH) using the oligonucleotide primers SEQ ID NO:69 and SEQ ID NO:70,the C. glutamicum chromosomal DNA as template and Pfu Turbo polymerase(Stratagene) with the aid of the polymerase chain reaction (PCR) bystandard methods, such as Innis et al. (1990) PCR Protocols. A Guide toMethods and Applications, Academic Press. The amplified fragment isflanked by an XhoI restriction cleavage site at its 5′ end and, at its3′ end, by a homologous region introduced via the oligo and homologousto Streptomyces coelicolor metH.

SEQ ID NO:69 5′-GAGACTCGAGGGAAGGTGAATCGAATTTCGG-3′ and SEQ ID NO:705′-GTCCCGGGGAGAACGCACGATTCTCCAAAAATAATCGC-3′

The resultant DNA fragment was purified with the GFX™PCR, DNA and GelBand Purification Kit (Amersham Pharmacia, Freiburg) following themanufacturer's instructions.

b) Starting from the Streptomyces coelicolor chromosomal DNA as templatefor a PCR reaction, a fragment of MetH was amplified with theoligonucleotide primers SEQ ID NO:71 and SEQ ID NO:72 with the aid ofthe GC-RICH PCR Systems (Roche Diagnostics, Mannheim) following themanufacturer's instructions. The amplified fragment is flanked at its 5′end by a region introduced via the oligo and homologous to the C.glutamicum HsDH promoter region.

SEQ ID NO:71 5′-GAATCGTGCGTTCTCCCCGGGAC-3′ and SEQ ID NO:725′-GTAGTTGACCGAGTTGATCACC-3′

The resulting approx. 1.4 kb DNA fragment was purified with the GFX™PCR,DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg)following the manufacturer's instructions.

c) In a further PCR reaction, the two fragments obtained above areemployed jointly as templates. Owing to the regions introduced with theoligonucleotide primers SEQ ID NO:71 and SEQ ID NO:70, which arehomologous to the respective other fragment, the two fragments annealwith each other during the PCR reaction and, owing to the polymeraseemployed, extend to form a continuous DNA strand. The standard methodwas modified in such a way that the oligonucleotide primers used, SEQ IDNO:69 and SEQ ID NO:72, were only added to the reaction mixture at thebeginning of the 2nd cycle.

The amplified, approximately 1.6 kb DNA fragment was purified with theGFX™PCR, DNA and Gel Band Purification Kit following the manufacturer'sinstructions. Thereafter, it was cleaved with the restriction enzymesXhoI and NotI (Roche Diagnostics, Mannheim) and separated by gelelectrophoresis. The approx. 1.6 kb DNA fragment was subsequentlyisolated from the agarose using the GFX™PCR, DNA and Gel BandPurification Kit (Amersham Pharmacia, Freiburg).

d) The metH 3′ region, which was still missing, was amplified startingfrom the Streptomyces coelicolor chromosomal DNA as template using theoligonucleotide primers SEQ ID NO:73 and SEQ ID NO:74 with the aid ofthe GC-RICH PCR system (Roche Diagnostics, Mannheim) following themanufacturer's instructions. The amplified fragment is flanked at its 3′end by an EcoRV restriction cleavage site introduced via the oligo.

SEQ ID NO:73 5′-CCGGCCTGGAGAAGCTCG-3′ and SEQ ID NO:745′-GAGAGATATCCCTCAGCGGGCGTTGAAG-3′

The resultant, approx. 2.2 kb DNA fragment was purified with theGFX™PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia,Freiburg) following the manufacturer's instructions. Thereafter, it wascleaved with the restriction enzymes NotI and EcoRV (Roche Diagnostics,Mannheim) and separated by gel electrophoresis. The approx. 2.2 kb DNAfragment was subsequently isolated from the agarose using the GFX™PCR,DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).

e) The vector pClik5MCS SEQ ID NO:63 (Example 1) was cleaved with therestriction enzymes XhoI and EcoRV (Roche Diagnostics, Mannheim), and a5 kb fragment was isolated following separation by electrophoresis,using the GFX™PCR, DNA and Gel Band Purification kit.

The vector fragment together with the two cleaved and purified PCRfragments were ligated with the aid of the Rapid DNA Ligation kit (RocheDiagnostics, Mannheim) following the manufacturer's instructions and theligation reaction was transformed into competent E. coli XL-1Blue(Stratagene, La Jolla, USA) as described by Sambrook et al. (MolecularCloning. A Laboratory Manual, Cold Spring Harbor, (1989)). Selection forplasmid-bearing cells was achieved by plating on kanamycin (20μg/ml)-containing LB agar (Lennox, 1955, Virology, 1:190).

The plasmid DNA was prepared by methods of, and using materials from,Quiagen. Sequencing reactions were carried out as described by Sanger etal. (1977) Proceedings of the National Academy of Sciences USA74:5463-5467. The sequencing reactions were separated and evaluated bymeans of ABI Prism 377 (PE Applied Biosystems, Weiterstadt).

The resultant plasmid pC Phsdh metH Sc (Streptomyces coelicolor) islisted as SEQ ID NO:79.

Sequence SEQ ID NO:79 comprises the following essential part-regions:

Type of Position sequence Description  6-155 Promoter HsDH  156-3752CDS¹⁾ MetH S. coelicolor 4153-4944 CDS Kanamycin resistance 5211-6071CDS replication origin (complement)²⁾ E. coli/Plasmid pMB ¹⁾codingsequence ²⁾on complementary strain

EXAMPLE 8 Transformation of Strain LU1479 LysC 311ile ET-16 with thePlasmid pC Phsdh metH Sc

Strain LU1479 lysC 311ile ET-16 (Example 5) was transformed with theplasmid pC Phsdh metH Sc (Example 7) by the method described (Liebl, etal. (1989) FEMS Microbiology Letters 53:299-303). The transformation mixwas plated onto CM plates supplemented with 20 mg/l kanamycin in orderto achieve selection for plasmid-containing cells. ResultantKan-resistant clones were picked and isolated. The methionineproductivity of the clones was studied in a shake-flask experiment (seeExample 6). Strain LU1479 lysC 311ile ET-16 pC Phsdh metH Sc producedsignificantly more methionine in comparison with LU1479 lysC 311 ileET-16.

1. A method for the fermentative production of at least onesulfur-containing fine chemical selected from L-methionine andS-adenosylmethionine, which comprises the following steps: a) fermentinga coryneform bacteria culture thereby producing the desiredsulfur-containing fine chemical, wherein the coryneform bacteriaexpresses at least one heterologous nucleotide sequence which codes fora protein with methionine synthase (metH) activity, wherein themetH-encoding sequence is selected from the group consisting of: 1) anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,or a complement thereof; and 2) a nucleic acid molecule comprising anucleotide sequence encoding a protein comprising an amino acid sequencewhich is at least 95% identical to the entire amino acid sequence of SEQID NO:2, or a complement thereof; and wherein said sequence is less than70% identical to the entire metH-encoding sequence from Corynebacteriumglutamicum ATCC 13032; b) concentrating the sulfur-containing finechemical in the medium or in the bacterial cells, and c) isolating thesulfur-containing fine chemical.
 2. The method of claim 1 wherein thesulfur-containing fine chemical comprises L-methionine.
 3. The method ofclaim 1, wherein the coding metH sequence is a DNA or RNA which can bereplicated in coryneform bacteria or is stably integrated into thechromosome of the coryneform bacteria.
 4. The method of claim 3, whereinthe coryneform bacteria is transformed with a plasmid vector comprisingat least one copy of the coding metH sequence under the control of aregulatory sequence.
 5. The method of claim 1, wherein the coding metHsequence is overexpressed.
 6. The method of claim 1, wherein thecoryneform bacteria further comprises the aspartate kinase-encoding genelysC that is overexpressed or mutated such that the aspartatekinase-encoding gene lysC encodes a protein having an isoleucine atamino acid residue 311 and the activity of the encoded protein isinfluenced by metabolic metabolites to a smaller extent.
 7. The methodof claim 1, wherein the coryneform bacteria is of the speciesCorynebacterium glutamicum.
 8. The method of claim 1, wherein thecoryneform bacteria is resistant to a methionine biosynthesis inhibitor.9. A method for producing an L-methionine-containing animal feedadditive from fermentation broths, which comprises the following steps:a) culturing and fermenting of an L-methionine-producing microorganismin a fermentation medium; wherein the microorganism expresses at leastone heterologous nucleotide sequence which codes for a protein withmethionine synthase (metH) activity, wherein the metH-encoding sequenceis selected from the group consisting of (a) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, or a complementthereof, and (b) a nucleic acid molecule comprising a nucleotidesequence encoding a protein comprising an amino acid sequence which isat least 95% identical to the entire amino acid sequence of SEQ ID NO:2,or a complement thereof; b) removing water from theL-methionine-containing fermentation broth; c) removing from 0 to 100%by weight of the biomass formed during fermentation; and d) drying thefermentation broth obtained according to b) and/or c), in order toobtain the animal feed additive in the desired powder or granule form.10. The method of claim 3, wherein the coding metH sequence isintegrated into the chromosome of the coryneform bacteria.